IT202100005438A1 - SCAFFOLD FOR BONE REGENERATION AND RELATED MANUFACTURING METHOD - Google Patents

Description

DESCRIPTION

of the patent for industrial invention entitled:

?SCAFFOLD FOR BONE REGENERATION AND ITS MANUFACTURING METHOD?

TECHNICAL FIELD

The present invention relates to a scaffold for bone regeneration.

In particular, the present invention belongs to the field of tissue engineering as a multidisciplinary sector which applies the principles of engineering and biology to create biological substitutes capable of restoring tissues, for example bone tissues, affected by a pathology, a trauma or following? Aging. Pi? precisely, the technical field of the present invention refers to a scaffold (i.e. a typically three-dimensional scaffold suitable for cellular support) loaded (even only in part) with functional material of cellular origin, the release of which promotes bone tissue regeneration, affected by a pathology, a trauma or following ageing.

STATE OF THE PRIOR ART

The bone tissue, the main constituent of the skeleton, is a highly specialized mineralized connective tissue, with important structural and metabolic functions. Through a process called ?bone remodeling?, the bone has the ability to intrinsic regeneration as part of the repair process in response to injury, as well as such as during skeletal development or ongoing remodeling throughout adult life. Sometimes, the physiological process of bone regeneration? compromised or insufficient, due for example to infections, osteonecrosis, trauma, tumours, intrinsic genetic diseases. In these cases, the bone graft? a widely used therapeutic strategy.

Although widely practiced, both autologous bone grafting (involving the transplantation of fresh cortical or trabecular bone, or a combination of both, from one site of the patient?s body to another) and allogeneic bone grafting (donor ) have limitations and problems.

Therefore, bone substitutes of biological and synthetic origin have been developed. Examples of synthetic materials are bioactive glasses, resorbable hydroxyapatite (HA), ?-tricalcium phosphate (TCP), and combinations thereof. They have been proposed for their similarity to bone mineral matrix, but, at present, there are no synthetic bone substitutes that have at least the same properties as bone mineral. biological or mechanical with respect to bone. On the other hand, materials of animal origin (xenografts) can provide structures similar to living tissue and thus induce specific cellular responses.

Materials combining natural and synthetic components have also been proposed, such as a biohybrid bone substitute composed of a bovine-derived bone matrix reinforced with poly(L-lactide-co-?-caprolactone) (PLCL). Scaffolds constructed with this material have recently been proposed as bone substitutes for oral, maxillofacial and dental implant surgery.

The above materials are used, in particular, to construct bone regeneration scaffolds.

By scaffold we mean a support, commonly three-dimensional and often porous, made with biocompatible and possibly bioabsorbable and/or biodegradable material, having the ability to to temporarily replace the natural tissue by favoring the mechanisms of differentiation, adhesion and proliferation of the cells loaded on it. During the production of the extracellular matrix by the cells, the scaffold tends to biodegrade and, simultaneously, to be replaced by the regenerated biological tissue, thus restoring the compromised function and at the same time avoiding surgical re-intervention for the removal of the prosthetic element.

? also known to load a scaffold with active substances, mostly? of cellular origin. The cells used can be of various types, for example: autologous, allogeneic, xenogenic and stem cells (both embryonic and adult). Among adult stem cells, mesenchymal stem cells (MSCs) currently represent a major resource in tissue engineering. In fact, MSCs can be easily isolated from the patient, in particular from bone marrow or adipose tissue, and expanded in culture up to a relevant physiological number. Furthermore, the MSCs have the ability? to differentiate into different cell lines such as, for example, osteoblasts, chondrocytes, myocytes and adipocytes, giving rise to various cellular tissues such as, for example, bones, cartilage, tendons, muscles and adipose tissue.

Current bone tissue engineering, based on three-dimensional scaffolds loaded with stem cells, including MSCs, has some limitations related, for example, to cell administration, such as rejection reactions, tumor induction and infection transmission. Furthermore, the manipulation of stem cells implies stringent control programs and elaborate protocols to standardize the production and conservation of large quantities of cells. of stem cells.

Furthermore, the fabrication of scaffolds loaded with stem cells is relatively complex and problems may arise in obtaining a suitable distribution and adhesion of the stem cells on the scaffold.

OBJECT OF THE INVENTION

? an object of the present invention is to provide a scaffold for the regeneration of bone tissue, and the related manufacturing method, which overcome the drawbacks described in the prior art.

In particular, a purpose of the invention ? that of providing a scaffold that allows an improved and controlled reconstruction of the damaged bone tissue. A further purpose of the invention ? that of providing a particularly effective, simple and inexpensive method for manufacturing said scaffold.

In accordance with these purposes, the present invention therefore relates to a scaffold for the regeneration of bone tissue and the related manufacturing method as defined in basic terms in the attached claims 1 and 8, respectively.

Further preferred features of the invention are indicated in the dependent claims.

Basically, the scaffold according to the invention ? consisting of a composite structure, formed by a bovine bone matrix and a biodegradable polymer, for example PLCL, integrating mesenchymal stem cell secretome (MSC-secretome), in particular lyophilized secretome (liosecretoma).

In fact, the loading of the scaffolds with the secretome allows for the creation of a bone substitute with improved resilience. osteoinductive and devoid of criticism? related to the use of stem cells as traditional engineered tissues require. Furthermore, compared to stem cells, the secretoma, particularly in the form of liosecretoma, lends itself to being prepared in advance to be ready for use in the case of acute pathologies that require timeliness.

The experimental results confirm that the scaffold according to the invention ensures rapid regrowth of bone tissue. and quality? of the new bone formation improved with respect to the prior art.

Also other features of the invention, implemented in the preferred embodiments, are particularly advantageous compared to conventional engineered tissues combining stem cells and scaffolds.

Preferably, the MSC-secretome used ? lyosecretome obtained by ultrafiltration and lyophilization, with mannitol (or other equivalent substance) as cryoprotectant.

According to a preferred embodiment of the invention, the lyosecretome ? loaded onto the bovine bone tissue scaffold by immersion and subsequent freeze-drying, resulting in the formation of a biologically active coating on the material surfaces.

The adsorption method? simple, direct and cheap to load the active substance (liosecretoma) on the scaffold.

To ensure effective loading and an equally effective release of the active substance, the formulation of liosecretoma is prepared with special excipients.

In particular, the solution of liosecretoma to be used for the adsorption on the scaffold is added with Lutrol? F127 (poloxamer 407) and NaCl.

Poloxamer 407 ? used to increase the wettability? of the support structure and facilitate the entry of the lisecretoma solution into the internal pores of the support structure so that the loading of the lisecretoma is homogeneous. In addition to favoring a homogeneous loading of the lisecretoma, poloxamer 407, like mannitol, stabilizes the proteins contained in the secretome, avoiding their denaturation. On the other hand, the introduction of NaCl prevents the crystallization of mannitol on the surface of the support structure during the freeze-drying process.

BRIEF DESCRIPTION OF THE DRAWINGS

Further characteristics and advantages of the present invention will become apparent from the description of the following non-limiting embodiments, with reference to the figures of the accompanying drawings, in which:

- figure 1 collects SEM images (Scanning Electron Microscopy) of reference scaffolds (non-functionalized: images a, b, c) and of scaffolds according to the invention (functionalized with lyosecretoma: images d, e, f, g, h, i) at increasing magnifications (30x and 100x);

- figure 2 ? a graph representing the release kinetics of functional substances (proteins and lipids) from scaffolds according to the invention (in vitro test; scaffolds immersed in PBS at pH 7.2 and room temperature);

- Figures 3A to 3D are magnified images (4x magnification) showing cell growth on a comparison scaffold, not functionalized with a lyosecretoma, and a scaffold according to the invention functionalized with a lyosecretoma: A, B respectively show the scaffold of comparison (CTRL SB) and the scaffold of the invention (CTRL SBlyos) without cell growth; C, D show the growth of SVF at 14 days on the comparison scaffold (SB SVF OM) and on the scaffold of the invention (SB SVF OM), respectively;

- Figures 4A to 4D are magnified images (20x magnification) showing cell growth, highlighted by H&E staining, after 60 days on a comparison scaffold, non-liosesecretoma functionalized (SB SVF OM), and on the scaffold according to the ?invention functionalized with liosecretoma (SBlyos SVF OM).

PREFERRED EMBODIMENT OF THE INVENTION

The scaffold according to the invention comprises a three-dimensional support structure consisting of a bone matrix of bovine origin, a synthetic polymer, in particular poly(L-lactide-co-?-caprolactone) (PLCL), and fragments of collagen, in particular type I collagen of animal origin.

In particular, the scaffold ? a composite scaffold having a shaped (i.e. consisting of a piece of bovine bone shaped into a predefined shape) or ground bovine bone matrix (i.e. consisting of a compacted aggregate of ground bovine bone) and coated with a polymeric material and containing fragments of collagen.

Scaffolds of this type, usable according to the invention, are currently marketed as Class III medical devices according to the 93/42/EEC directive, in particular under the commercial name of SmartBone?, and have recently been proposed as bone substitutes for oral surgery, maxillofacial surgery, dental implantology, orthopedic and oncological applications. Details on these scaffolds are available in the literature, for example in:

WO2010070416A1;

?Reconstruction of the Zygomatic Bone with SmartBone?: Case Report? - Journal of Biological Regulators and Homeostatic Agents 2015, 29, 42-47;

?Clinical and Histological Evaluation of Socket Preservation Using SmartBone?, a Novel Heterologous Bone Substitute: a Case Series Study? - Oral Implantology 2018, XI);

?Composite Xenohybrid Bovine Bone-Derived Scaffold as Bone Substitute for the Treatment of Tibial Plateau Fractures? - Appl. Sci. 2019, 9, 2675; doi:10.3390/app9132675;

?A Preliminary Study on the Mechanical Reliability and Regeneration Capability of Artificial Bone Grafts in Oncologic Cases, With and Without Osteosynthesis? - J. Clin. Med. 2020, 9, 1388; doi:10.3390/jcm9051388.

Advantageously, the scaffold ? treated to integrate collagen fragments containing RGD (arginylglycylaspartic acid) to promote vitality cell phone, l?hydrophobicity? of the matrix and the biocompatibilit? in general, as illustrated in Pertici G. et al. ?Composite Polymer-coated Mineral Scaffolds for Bone Regeneration: from Material Characterization to Human Studies? ? J. Biol. Regul. homeost. Agents, 2015; 29(3 Suppl 1):136-48; and in Cingolani A. et al. ?Improving Bovine Bone Mechanical Characteristics for the Development of Xenohybrid Bone Grafts? - Current Pharmaceutical Biotechnology 2018, 19, 1005-1013, doi:10.2174/1389201020666181129115839.

According to the invention, the support structure ? loaded and then functionalized with lyophilized secretome (liosecretome), in particular secretome of mesenchymal stem cells, defining a functional material conveyed by the support structure.

The support structure can have various shapes, sizes and geometries, also depending on the specific use for which the scaffold is intended.

The scaffold can be made according to one of the known techniques available, for example through a 3D printing process; or can? be shaped from a piece of bovine bone treated with the polymer and the collagen fragments; or by aggregating and compacting ground bovine bone together with the polymer and collagen fragments in a mold.

Subsequently, the scaffold is functionalized with the secretome by adsorption, by immersing the scaffold in a solution containing the secretome.

Is the secretome present on the support structure? in particular mesenchymal stem cell secretoma (MSC-secretoma).

Advantageously, the MSC-secretoma ? isolated and purified from cultured mesenchymal stem cell (MSC) supernatants by a combined ultrafiltration and freeze-drying process described in patent application WO2018/078524.

In summary, the process of obtaining the secretome includes the steps of:

(i) withdrawing a supernatant from cultured mesenchymal stem cells, comprising both a soluble fraction (essentially proteins) and an insoluble fraction in particulate form (vesicles),

(ii) dialyze or ultrafiltrate the biological fluid using a membrane having a cut-off value equal to or less than 500,000 Daltons and a dialysis or ultrafiltration fluid,

(iii) adding a cryoprotectant to the obtained dialyzed or ultrafiltered solution, e

(iv) freeze-drying the resulting solution.

The ultrafiltered supernatant is added with a cryoprotectant, preferably mannitol, which allows to prevent unwanted effects, such as the disintegration of the extracellular vesicles and the instability of proteins, induced by flash freezing and drying during the freeze-drying process. At the end of the process, a lyophilized powder is obtained, called lyosecretome (lyophilized secretome), which contains the secretome obtained from mesenchymal stem cells (MSCs).

In a preferred embodiment, the lyosecretome is loaded onto the support structure by immersing the support structure in a solution containing the lyosecretome, followed by lyophilization, so that the surfaces of the support structure are coated with the lyosecretome. Specifically, two excipients are added to the liosecretoma solution, such as Lutrol? F127 (poloxamer 407) and NaCl. Poloxamer 407 ? used to increase the wettability? of the support structure and facilitate the entry of the lisecretoma solution into the internal pores of the support structure so that the loading of the lisecretoma is homogeneous. In addition to favoring a homogeneous loading of the lisecretoma, poloxamer 407, like mannitol, stabilizes the proteins contained in the secretome, avoiding their denaturation. On the other hand, the introduction of NaCl prevents the crystallization of mannitol on the surface of the support structure during the freeze-drying process.

EXAMPLES

Some experimental examples are given below which describe the manufacture and effectiveness of scaffolds according to the invention.

1. Preparation of liosecretoma

The lyosecretoma (lyophilized secretome of mesenchymal stem cells) ? been prepared according to the modalities? indicated above, substantially as reported in WO2018/078524.

In particular, ? Lyosecretome obtained from adipose-derived cells (AD-MSCs) harvested from adipose tissues was used. Were the cells cultured using standard. The release of secretome from cells ? was obtained by culturing cells in DMEM/F12 without platelet lysate for 48 h; conditioned media were collected after 9, 24, 33 and 48 hours and pooled. The MSCs were detached with trypsin-EDTA and tested for viability. cell phone and compliance with all necessary requirements for clinical use.

The conditioned media were centrifuged at 3500 g/min for 10 min to eliminate cellular debris and apoptotic bodies, obtaining supernatants which were collected and ultrafiltered by tangential flow filtration. Both free soluble proteins and vesicles produced by MSCs were retained.

Samples were initially concentrated at 0.5 ? 10<6 >cell equivalents per mL (calculated by dividing the total number of cells by the mL of concentrated and purified supernatant) and then diafiltered using sterilized ultrapure water.

In the concentrated and purified secretome ? dissolved state mannitol (final concentration 0.5% w/v); the resulting solution? been frozen at -80?C and freeze-dried at 8 ? 10<-1 >mbar and -50?C for 72 hours.

The lyosecretoma obtained ? been stored at -20?C until use. Each mg of liosecretoma corresponds to 0.1 ? 10<6 >cell equivalents (calculated by dividing the total number of cells used for production and the milligrams of lyosecretoma obtained).

? was determined in the obtained lyosecretoma a protein content of 30.72 ? 2.139 ?g and a lipid content of 1.62 ? 0.0329 ?g per mg lyophilized powder (mean value ? standard deviation obtained from three independent replicates from the same lyosecretoma lot).

2. Preparation of scaffolds functionalized with lyosecretoma

Scaffolds were made according to what was previously reported in the literature (in particular in WO2010070416A1 and in ?Composite polymercoated mineral scaffolds for bone regeneration: from material characterization to human studies? ? J. Biol. Regul. Homeost. Agents, 2015; 29(3 Suppl 1):136-48).

Yes ? used bovine-derived spongy bone certified for human use and free from bovine spongiform encephalopathy. The scaffolds were then obtained by reinforcing the bovine bone-derived matrix with a mixture of PLCL and RGD-containing collagen fragments. After the final double layer packaging, the product is ? been sterilized with ethylene oxide.

Subsequently, the scaffolds were functionalized with lyosecretoma, particularly by adsorption.

Each sample scaffold (size 1 cm ? 1 cm ? 0.3 cm) ? was loaded with 16,000 cell equivalents of lisecretoma.

Were the samples individually immersed in an aqueous solution containing lyosecretoma (1.7 mg/mL), Lutrol? F127 (0.1% w/v) and NaCl (0.1% w/v) for 1 hour at 4°C.

Lutrol? F127 ? been added as a surfactant to increase wettability? of the support structure and allow cos? a loading pi? homogeneity of the lyosecretoma, allowing the solution containing the lyosecretoma to penetrate inside the support structure. NaCl ? was added to reduce the crystallization of mannitol during the freeze-drying process.

Figure 1 shows the SEM morphological investigation of: - scaffolds according to the invention loaded with lyosecretoma with the addition of poloxamer 407 (images d, e, f);

- scaffolds according to the invention loaded with lyosecretome with the addition of poloxamer 407 and NaCl (images g, h, i);

- comparison scaffolds, not loaded with liosesecretoma (images a, b, c).

SEM images were taken with increasing magnifications (the first two images of each group at 30x magnification, the third at 100x magnification).

Morphological characterization revealed the deposition of a liosesecretoma coating on the surfaces of the support structure material (images d, e, f). The external pores were almost completely closed cos? like the internal ones (with some exceptions in the center, image e).

The addition of NaCl as an excipient to prevent the crystallization of mannitol is effective. proved to be advantageous also from the point of view of the deposition of the lyosecretoma. In fact, using a formulation with poloxamer 407 and NaCl ? a uniform deposition of lisecretoma was observed on the surface of the support structure (image g), with a complete closure of the external pores as well as of the central pores (images h, i).

The samples were then frozen at -80?C and freeze-dried at 8?10<-1 >mbar and -50?C for 24 h.

For determination of protein/lipid loading, samples were dispersed in deionized water under magnetic stirring for 96 h. The solutions obtained were tested to determine the protein and lipid content. The average content of proteins and lipids ? result equal to 657.25 ? 6.662 ?g and 18.44 ? 2.452 ?g, respectively.

3. Release of active substance

The scaffold samples were tested by immersion in sulphate buffered saline (PBS) at pH 7.2 to evaluate the speed? release of functional substances, in particular proteins and lipids, incorporated in the lyosecretoma.

The release profiles of proteins and lipids are shown in Figure 2. Cumulative release from the scaffolds showed an initial concentrated release, in 30 minutes, of 57% for proteins and 86% for lipids. 100% proteins and lipids ? was released after 24 hours. This quick release ? presumably due to the components of the formulation (mannitol, poloxamer 407 and NaCl) which being hydrophilic compounds easily recall the PBS in the scaffold and dissolve.

The complete release of the secretome from the scaffold after the times indicated above? been confirmed by via SEM investigation.

4. Stromal vascular fraction (SVF) tissue growth

For comparison purposes, ? The tissue growth of the stromal vascular fraction was evaluated on scaffolds according to the invention, functionalized with a lisecretome, and reference scaffolds, completely analogous but not functionalized with a liosesecretoma.

Growth ? was evaluated at 14 days and 60 days. Figures 3A and 3B are initial images, before cell growth, of a non-functionalized scaffold and a functionalized scaffold with lyosecretoma, respectively. ? H&E staining was performed, showing the trabecular structure of the cell-free scaffolds (Figure 3A and 3B, respectively) and colonization of the scaffold by SVF, showing initial new tissue formation at 14 days (Figure 3C and Figure 3D) , mainly on the sample functionalized with lisecretoma.

After 60 days, the presence of new bone tissue was evident on both the comparison scaffold and the scaffold of the invention (functionalized with a lyosecretoma) (Figures 4A-D). In all samples, SVF colonized and formed new tissue, starting at the periphery (Figure 4A) of the scaffold and filling the bone gaps (Figure 4B). Although the area of new tissue formation was detectable on both scaffolds, growth was more pronounced. labeled on the functionalized sample, as shown in Figures 4C and 4D, respectively.

The increased formation of well-organized bone tissue ? therefore attributable to the presence of the lyosecretoma.

To further confirm this result, ? Collagen 1 (COLL-1), osteocalcin (OCN) and transforming growth factor beta (TGF?) staining was performed on both non-functionalized and lyosecretoma-functionalized scaffolds. ? The expression of these proteins by cells grown on the scaffolds was checked at 60 days. OCN and TGF? were highly expressed, while COLL-1 was weakly positive. However, COLL-1 markedly colored the new tissue on the functionalized scaffold.

All the results confirm that although in all the scaffolds tested (functionalized and not) the formation of bone tissue is observed, the scaffolds according to the invention show a greater and more? fast growth of bone tissue.

Ultimately, the experimental evidence confirms that a scaffold consisting of a bovine matrix and a biodegradable polymer (PLCL) and containing fragments of collagen and functionalized with lyosecretome promotes faster bone growth. rapid and effective compared to the same non-functionalized scaffold.

After depositing SVF on the functionalized and non-functionalized scaffolds, the tissue ingrowth ? was monitored at 14 and 60 days, showing growing areas of new tissue over time. After 14 days, ? Increased cell proliferation was observed on the lisecretoma-functionalized scaffold, where initial cellular filling of the gaps between the trabeculae was detectable. On the comparison scaffolds (without lisecretoma), on the other hand, the process is been more slow and tissue formation ? was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. However, the scaffolds of the invention (with liosecretoma) showed a higher expression of osteoblast markers and a higher amount of osteoblasts. of newly formed trabeculae compared to the comparison scaffolds. Quantification analysis of newly formed mineralized tissue demonstrated that lisecretoma induces bone formation more effective. Furthermore, the immunohistochemical analysis performed at 60 days allowed to observe the presence of highly organized bone tissue on the scaffolds with lyosecretoma, indicative of cells stimulated to grow and differentiate.

Claims (11)

1. Scaffold for bone regeneration, comprising a support structure made with a composite hybrid formed by a bovine bone-derived matrix reinforced with at least one polymer and containing collagen fragments; the support structure being functionalized with secretome. 2. Scaffold according to claim 1, wherein the secretome is mesenchymal stem cell secretome. 3. Scaffold according to claim 1 or 2, wherein the secretome is lyosecretoma, ie? lyophilized secretome. 4. Scaffold according to one of the preceding claims, wherein the secretome contains both a soluble fraction, in particular comprising proteins, and a particulate insoluble fraction, in particular extracellular vesicles. 5. Scaffold according to one of the preceding claims, wherein the secretome contains proteins and lipids. 6. Scaffold according to one of the preceding claims, wherein the bovine bone derivation matrix is reinforced with poly(L-lactide-co-?-caprolactone) (PLCL). 7. Scaffold according to one of the preceding claims, wherein the collagen fragments are of animal origin. 8. Method of manufacturing a scaffold for bone regeneration according to one of the preceding claims, comprising the steps of: manufacturing the support structure by 3D printing; and loading the support structure with the secretome. The manufacturing method according to claim 8, wherein the step of loading the support structure with the secretome is carried out by adsorption of the secretome on the support structure. The manufacturing method according to claim 9, wherein the secretome is loaded onto the support structure by immersing the support structure in a solution containing the secretome, and subsequent freeze-drying. 11. A manufacturing method according to claim 9 or 10, wherein the secretome is in solution with poloxamer 407 and/or NaCl.